Summary

في تقييم فيفو من القوارض المتصورة الطفيل وأقسومة الغزو من قبل التدفق الخلوي

Published: April 05, 2015
doi:

Summary

ويغزو الطفيلي الملاريا ومكررات داخل خلايا الدم الحمراء. تقييم دقيق للغزو أقسومة والطفيل هو بالتالي حاسما في تقييم مسار عدوى الملاريا. نحن هنا وصف التدفق الخلوي بروتوكول أساس لقياس هذه المعايير في نموذج الفأر من الملاريا.

Abstract

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Introduction

The clinical symptoms associated with malaria occur during the Plasmodium parasite’s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs.

Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia.

We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

Protocol

وأجريت جميع الإجراءات وفقا لسياسات جامعة ماكواري ويتفق مع المجلس الوطني للصحة والبحوث الطبية (NHMRC) رمز الاسترالي من الممارسة. تم إجراء العمل في إطار أخلاقيات اتفاق لا ARA 2012/017 الموافقة والحصول عليها من لجنة الأخلاقيات الحيوانية في جامعة ماكواري. أجريت جميع التجارب على الفئران SJL …

Representative Results

قياس الطفيل. لقياس الطفيل، ينبغي أولا أن يتم اختيار خلايا الدم، والضوضاء، والحطام والصفائح الدموية استبعاد، على أساس FSC / خصائص التعاون بين بلدان الجنوب (الشكل 2A). اعتمادا على عداد الكريات المستخدمة، وينبغي بعد ذلك يتم اخت…

Discussion

وصفناها طريقة لقياس كل من الغزو الطفيل وأقسومة العينات في الجسم الحي. من حيث قياس الطفيل، وهذه الطريقة يوفر ميزة على الطرق السابقة 10-13 في ذلك HJ-كرات الدم الحمراء يمكن تمييزها عن pRBCs، مما يقلل من عدد من الأحداث الإيجابية الكاذبة. بينما HJ-كرات الدم الحمراء وعا…

Disclosures

The authors have nothing to disclose.

Acknowledgements

ونحن نعترف تمويل الدعم من مجلس البحوث الصحية والطبية الوطنية (منح APP605524، 490037 1047082 و)، ومجلس الأبحاث الأسترالي (منح DP12010061)، والاستراتيجية البنية التحتية البحوث التعاونية الوطنية في أستراليا وصندوق الاستثمار التعليم من قسم الابتكار والصناعة والعلوم والبحوث. حزب الرابطة الاسلامية هي المستفيدة من جائزة الدراسات العليا الاسترالية.

Materials

bisBenzimide H 33342 trihydrochloride Sigma-Aldrich B2261 Hoechst 33342. Store a 4mM stock solution at -20 °C in distilled water
Hoechst 34580 Sigma-Aldrich 63493 Store a 2mM stock solution at -20 °C in distilled water
JC-1 Dye Life Technologies T-3168 Store small aliquots of 6mM stock solution at -20 °C in DMSO
Anti-Mouse CD45 APC-eFluor 780 eBioscience 47-0451-80 Clone 30-F11
Anti-Mouse CD71 PerCP-eFluor 710 eBioscience 46-0711-80 Clone R17217
Atto 633 NHS ester Sigma-Aldrich 1464 Atto 633-NHS. Store a 2mg/ml stock solution at -20 °C in DMF
EZ-Link Sulfo-NHS-LC-Biotin Thermo Fisher Scientific 21335 Biotin-NHS. Store a 25mg/ml stock solution at -20 °C in DMF
Streptavidin PE-Cyanine7 eBioscience 25-4317-82 Streptavidin PE-Cy7
Heparin Sigma-Aldrich H478
35µM filter cap tubes Becton Dickinson 352235
Flow cytometer: BD LSRFortessa Becton Dickinson
Flow cytometer: BD FACSAria II Becton Dickinson
Flow cytometer: BD Influx Becton Dickinson
Flow cytometer: CyAn ADP Analyzer Beckman Coulter

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Cite This Article
Lelliott, P. M., McMorran, B. J., Foote, S. J., Burgio, G. In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry. J. Vis. Exp. (98), e52736, doi:10.3791/52736 (2015).

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